A Bionic‐Homodimerization Strategy for Optimizing Modulators of Protein–Protein Interactions: From Statistical Mechanics Theory to Potential Clinical Translation

Abstract Emerging protein–protein interaction (PPI) modulators have brought out exciting ability as therapeutics in human diseases, but its clinical translation has been greatly hampered by the limited affinity. Inspired by the homodimerize structure of antibody, the homodimerization contributes hugely to generating the optimized affinity is conjectured. Herein, a statistical‐mechanics‐theory‐guided method is established to quantize the affinity of ligands with different topologies through analyzing the change of enthalpy and the loss of translational and rotational entropies. A peptide modulator for p53‐MDM2 termed CPAP is used to homodimerize connecting, and this simple homodimerization can significantly increase the affinity. To realize the cellular internalization and tumor accumulation, DimerCPAP and MonoCPAP are nanoengineered into gold(I)‐CPAP supermolecule by the aurophilic interaction‐driven self‐assembly. Nano‐DimerCPAP potently suppressed tumor growth in lung cancer allograft model and a patient‐derived xenograft model in more action than Nano‐MonoCPAP, while keeping a favorable drug safety profile. This work not only presents a physico‐mechanical method for calculating the affinity of PPI modulators, but also provides a simple yet robust homodimerization strategy to optimize the affinity of PPI modulators.


Experimental Section General remarks
All synthetic peptide sources were obtained from CS Bio (Shanghai) Ltd. All other chemicals used in this study were purchased from Sigma-Aldrich unless otherwise specified.
Acetonitrile and water (HPLC grade) were purchased from Fisher Scientific Ltd. All products were used as received without further purification.

Synthesis of Mono CPAP or Dimer CPAP
All peptides were synthesized on appropriate resins on an CS bio 336X automated peptide synthesizer using the optimized HBTU activation/DIEA in situ neutralization protocol developed by an HBTU/HOBt protocol for Fmoc-chemistry SPPS. After cleavage and deprotection in a reagent cocktail containing 88% TFA, 5% phenol, 5% H 2 O and 2%TIPS, crude products were precipitated with cold ether and purified to homogeneity by preparative C18 reversed-phase HPLC.
The molecular masses were ascertained by electrospray ionization mass spectrometry (ESI-MS). Origin program. Data points at saturation were used to calculate a mean baseline value, which was then subtracted from each data point.

Fluorescence Polarization (FP)-Based Binding and Competitive Binding Assay
To perform the FP binding assay, fluorescein isothiocyanate (FITC) was conjugated to MDM2 via its N-terminal amino group. To perform the FP competitive binding assay, fluorescein isothiocyanate (FITC) was conjugated to 15−29 p53 via its N-terminal amino group. The resultant products MDM2-FITC or 15−29 p53-FITC was HPLC-purified and lyophilized. MDM2 was synthesized and purified as previously described. [1] Fluorescence polarization-based binding assay was then performed as described previously [2] , and the readings were taken using a fluorescence microplate reader (Tecan M2000, λex = 470 nm; λem = 530 nm.). Mono CPAP or Dimer CPAP peptide was serially 2-fold diluted in 10 mM Tris-HCl buffer (pH 7.0) containing 150 mM NaCl and 1 mM EDTA, and subsequently incubated with 200 nM MDM2-FITC or 15−29 p53-FITC/MDM2 for 2 h in a total volume of 150 μL per well. Kd and IC 50 values were calculated by nonlinear regression as described previously. [1] Synthesis of Nano-Mono CPAP or Nano-Dimer CPAP First, 2 mg of Mono CPAP or Dimer CPAP peptide was completely dissolved in a solution containing 500μL ethanol and 1.25mL ddH 2 0. After that, an aqueous solution of tetrachloroauric acid (HAuCl 4 ·XH 2 O, 1 mL, 10 mM) was mixed with 500μL NH 2 -PEG-SH (MW: 2000, 4 mg/ml in deionized water) and 2.25 ml HEPES (100mM, pH 7.0). Then it mixed with the prepared solution containing 2.25 ml deionized water and 2.25ml HEPES (100mM pH 7.0), sonicate for 10 min. Finally, removed the excess reactants by dialysis tubing (cutoff, 10 KDa) and washed twice by distilled water. Finally, we obtained Nano-Mono CPAP and Nano-Dimer CPAP.

Physicochemical properties of Nano-Mono CPAP or Nano-Dimer CPAP
The morphology and lattice structure were observed on high-resolution transmission electron microscopy (HRTEM), which was performed on an Talos F200X. One portion of the pellet was placed onto a carbon-coated copper grid for imaging with high-resolution transmission electron microscopy (HRTEM). The hydrodynamic size distribution was obtained from the dynamic light scattering (DLS) measurement (Malvern Zetasizer Nano ZS system). For Zeta potential measurement, Nano-Mono CPAP or Nano-Dimer CPAP was incubated with PBS at pH 7.4.

Cell culture and cell cycle analysis
Human NSCLC cell lines NCI-H1650 were obtained from the Chinese Academy of Science Cell Bank (Shanghai, China). NCI-H1650 was cultured in RPMI-1640 medium, with all recommended supplements. NCI-H1650 was maintained at 37 °C in a humidified incubator with 5% CO 2 .
Cells were plated in 6-well plates at a density of 2×10 5 cells. After under serum starved condition, cells were treated with control, Nano-Mono CPAP or Nano-Dimer CPAP respectively. After 48 hours, the cells were separately collected, Washed twice with cold PBS, resuspended in 500 μL of PBS, added 4.5 mL of 70% ethanol while shaking and mixed, and fixed at -20 °C for 12 hours.
After PBS washing, centrifugation, resuspending in 500 μL of 50 μg/mL ethidium bromide (PI), 100 ug/mL RNase A, 0.2% Triton X-100 in PBS, incubating at 4°C for 30 minutes in the dark, and using flow cytometry analysis. FlowJo software was used for analysis of cell cycle.

Transcriptome analyses
NCI-H1650 cells were seeded in 6-well plates and allowed to attach overnight. Cells were incubated with control, Nano-Mono CPAP or Nano-Dimer CPAP at a concentration of 2×10 5 particles/cell in RPMI-1640 medium with 10% FBS for 24 h at 37 °C. Total RNA from cells were isolated using Trizol and the quantity and quality of the resulting RNA was measured using a 2100 Bioanalyzer chip.

In vivo antitumor efficacy of Nano-Dimer CPAP
Animal studies were performed according to the protocols approved by the Institution Guidelines and were approved by the Laboratory Animal Center of Xi'an Jiaotong University.
ALL mice were purchased from the Laboratory Animal Center of Xi'an Jiaotong University. The mice were housed under standard specific pathogen-free conditions with a 12h-12h light-dark cycle.

Subcutaneous tumor-bearing mice model
C57BL/6 mice (aged 6-7 weeks) were age-matched for tumour inoculation. LLC cells (1 ×10 6 cells/site) were implanted subcutaneously into hip of C57BL/6 mice. When the tumors reached average volume of ~ 75 mm 3 , mice were selected randomly into control group, Nano-Mono CPAP (2.5mg/Kg) or Nano-Dimer CPAP (2.5mg/Kg) group respectively (6 mices per group). Treatment was administered via intraperitoneal injection every other day. The body weight and condition of mice were monitored daily. In addition, tumor length and width were measured with calipers daily, and tumour volumes were calculated using the following equation: 1/2× l × w 2 .
The humane end points were determined on the basis of the level of animal discomfort and tumour sizes.

Patient-derived xenografts mice model
The homogenized tumor tissue with non-necrotic was cut into about 5 mm pieces and implanted into the right upper limb of each Female Balb/c nude mice (4-5 weeks) while under anesthesia. After two weeks, mice were selected into different groups. Then we treated tumor-burdened mice with PBS, Nano-Mono CPAP (2.5mg/Kg) or Nano-Dimer CPAP (2.5mg/Kg) through intravenous injection every other day, and monitored the tumor growth for 15 days. Daily monitoring of mice was the same as above subcutaneous tumor-bearing mice.

H&E and immunohistochemistry
All sections used for histological analysis were 4-μm thick. The tumors and the major organs (heart, liver, spleen, lung, and kidney) after different treatments were harvested for H&E staining.
Moreover, the tumors were sectioned for cell apoptosis analysis by TUNEL apoptosis assay.

Statistics
All the experimental data were measured in triplicate at least and are presented as mean ± standard deviation unless otherwise mentioned. Statistical variance of two comparison groups was performed at a significance level of p < 0.05 based on a Student's t-test. comparisons of more than two groups were calculated using a one-way analysis of variance (ANOVA), or log-rank test where necessary.

Derivation of the dissociation constant based on statistical mechanics
Suppose that a ligand (L) can bind to a receptor (R). The dissociation constant d K for the chemical reaction between receptors and ligands is defined as  The partition function R q or L q of a free receptor or ligand involves three translational degrees of freedom (described by x, y, and z) and three rotational degrees of freedom (described by  ,  , and  ). The degrees of freedom of the partition function RL q of a single R-L bond is the sum of those of R q and L q . Then, the partition functions I q of a single molecule are where the configurational space where the configurational space (S15) Using the chemical potential and the equilibrium condition of the reaction one can obtain (S18) The concentration is defined as [] , and then the dissociation constant in Eq. (S1) is re-expressed as (S19) Using Eqs. (S12)-(S14), the difference of the free energy between a bond, a free receptor and a free ligand is ln ln where the terms related to the kinetic energy is cancelled. Equation (S20) indicates that the kinetic energy plays no role in G  , although it affects the absolute free energy of molecules.
Substituting Eq. (S20) into Eq. (S19) leads to The configurational integrals of an unbound receptor or ligand are respectively where b U is the binding enthalpy of a receptor and a ligand,